AGK in cardiomyocyte and cardiomyopathy

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PROJECT SUMMARY Sengers syndrome is a recessive mitochondrial disease, with over 85% of patients diagnosed with pediatric cardiomyopathy, which can lead to heart failure and premature death. Loss-of-function mutations in acylglycerol kinase (AGK) have been identified as causes of Sengers syndrome, highlighting the significant role of AGK in heart function. However, the specific role of AGK in cardiomyocytes (CMs), or molecular mechanisms by which AGK loss of function leads to cardiomyopathy, has not yet been identified. Initially described as a multi-substrate lipid kinase in mitochondria, AGK phosphorylates mono- and di- acylglycerol to produce lysophosphatidic acid (LPA) and phosphatidic acid (PA), respectively. Given the crucial cardiac functions of LPA and PA, which act as signaling molecules or feed into the synthesis of phospholipids (such as cardiolipin), the pathological etiology of Sengers syndrome was originally linked to the lipid kinase activity of AGK. However, evidence of reduced levels of LPA, PA, or other phospholipids in patients or experimental models of Sengers syndrome is currently lacking. Consequently, it remains unclear whether the cardiomyopathy in Sengers syndrome is a result of a deficiency in AGK’s kinase function. Independent of its kinase activity, in vitro studies have identified AGK as a component of the mitochondrial TIM22 complex, regulating the import of mitochondrial carrier proteins, suggesting an alternative pathogenic mechanism for Sengers syndrome. In addition, recent studies found that a small portion of AGK protein present in the non- mitochondrial fraction regulates signal transduction via both kinase-dependent and -independent mechanisms. However, it is unclear which aspect(s) of AGK function, such as kinase activity, the TIM22 component function, or other as yet unidentified roles, are essential for maintaining normal cardiac structure and function. To investigate the cardiac role of AGK, we generated Agk global knockout (gKO) mice and CM-specific knockout (cKO) mice. Preliminary data indicate that Agk gKO and cKO mice develop similar cardiomyopathy and premature lethality, suggesting that the cardiac phenotype in Agk gKO mice is due to loss of Agk in CMs. Lipidomic analyses revealed a 40% decrease in LPA levels in Agk gKO hearts, while levels of other lipids, including PA and cardiolipin, were unchanged compared to wildtype hearts. Moreover, protein levels of Tim29 and Tim22, as well as a subset of TIM22 substrates, were reduced in Agk gKO hearts. AGK deletion also led to protein aggregation in the heart, suggesting a previously unknown function of AGK in protein homeostasis. The foregoing findings lead us to the hypothesis that AGK plays an essential role in CMs through its multifaceted functions, and that both kinase-dependent and -independent roles of AGK are critical for cardiac function. Accordingly, our specific aims are: 1) To determine the mechanism(s) by which AGK deficiency causes cardiomyopathy; 2) To dissect kinase-dependent and TIM22 complex-dependent functions of AGK.